Methods of identifying synthetic transcriptional and translational regulatory elements, and compositions relating to same
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EXAMPLE 1
Selection of Synthetic Transcriptional Regulatory Elements
[0118]This example describes the preparation of a vector useful for selecting transcriptional regulatory elements and the identification and characterization of synthetic transcriptional regulatory elements.
[0119]A promoter element proviral vector library was constructed using the retroviral-mediated EGFP / FACS selection strategy for synthetic promoter elements according to the disclosed methods. A library of promoter elements (random 18 mers; Ran18) was constructed in the proviral selection vector, which was packaged into retroviral particles in COS1 cells. The retroviral particles were harvested and used to infect target cells, which were then treated for 3 days with puromycin to kill uninfected or poorly expressing cells. The surviving cells were subjected to FACS analysis and the most highly fluorescent cells collected. Genomic DNA was prepared from these cells and the regulatory oligonucleotides were recovered by...
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EXAMPLE 2
Validation of Synthetic Regulatory Element Selection Method
[0164]This example demonstrates the disclosed synthetic regulatory element selection method can be used routinely to screen libraries of oligonucleotides and can consistently identify synthetic transcriptional regulatory elements.
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[0165]The retrovirus vector MESVR / EGFP* / IRES / pacPro(ori) (SEQ ID NO: 1; see Example 1B) was used to screen a second library of Ran 18 sequences using the synthetic promoter construction method (SPCM). More than 100 DNA sequences that showed increased promoter activity (4 to 50-fold) in the neuroblastoma cell line Neuro2A were identified. The DNA sequences of selected synthetic promoters were determined and database search using the RIGHT software package, which allowed simultaneous comparison of a database of active Ran 18 elements to existing databases such as TransFac. The search revealed a predominance of eight motifs—AP2, CEBP, GRE, Ebox, ETS, CREB, AP1, and SP1 / MAZ; about 5 to 10% of the active DNA sequences were not represented in known transcription factor databases and appeared to be novel. The most active of the selected synthetic promoters contained composites of pairs, triples, or quadruples of these motifs. Assays of DNA binding and promoter activity of three exemplary m...
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